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Abstract

Label-free imaging of rapidly moving, sub-diffraction sized structures has important applications in both biology and material science, as it removes the limitations associated with fluorescence tagging. However, unlabeled nanoscale particles in suspension are difficult to image due to their transparency and fast Brownian motion. Here we describe a novel interferometric imaging technique referred to as Magnified Image Spatial Spectrum (MISS) microscopy, which overcomes these challenges. The MISS microscope provides quantitative phase information and enables dynamic light scattering investigations with an overall optical path length sensitivity of 0.95 nm at 833 frames per second acquisition rate. Using spatiotemporal filtering, we find that the sensitivity can be further pushed down to 10−3-10−2 nm. We demonstrate the instrument’s capability through colloidal nanoparticle sizing down to 20 nm diameter and measurements of live neuron membrane dynamics. MISS microscopy is implemented as an upgrade module to an existing microscope, which converts it into a powerful light scattering instrument. Thus, we anticipate that MISS will be adopted broadly for both material and life sciences applications.

Figures (8)

Fig. 1 MISS microscope. f1 = 60 mm, f2 = 150 mm, f3 = 0.3 mm. L3 and L2 form a 4-f system that magnifies the Fourier transform of the zero-order by a factor of 500. For the L3-L2 imaging system, image formation at both high and low spatial frequencies is shown to illustrate the magnification process.

Fig. 3 Analysis of spatiotemporal stability of the MISS microscopy system. (a) A 256 x 1500 pixels no-sample OPD image with color bar in nm. (b) The histogram of the noise OPD stack acquired at 833 fps. (c) Plot showing the noise content at each spatial and temporal frequency component along three different planes in 3d frequency space. Color bar is in log scale with units of nm2(rad/μm)2(rad/s). (d) Band-pass filtering over the spatio-temporal bands shown in (c) results in noise values 2-3 orders of magnitude less than the total noise of 0.95 nm.

Fig. 6 Measurement of neuron membrane dynamics and surface tension σ before and after high K+ stimulation. (a-b) Fluorescence images of a neuron before and after stimulation, respectively. The enhanced fluorescence signal after stimulation reports on the intracellular Ca+2 ion concentration. (c-d) Representative phase images of the neuron. (e-f) Temporal standard deviation of the OPD time-lapse overlaid over the corresponding phase image. (g) DPS analysis on four different cells before high K+ stimulation. Linear fitting of the average dispersion curve yields the mean surface tension σof the neuron membrane. (h) Dispersion curve for a no-sample, i.e., background (BG) region. (i) DPS analysis on the same four cells after high K+ stimulation. Linear fitting of the average dispersion curve was used to extract mean surface tension σof the neuron membrane.

Fig. 7 (a) and (b) Average raw intensity images obtained for MISS and DPM, respectively, by temporally averaging 1024 frames. (c) Histograms of the intensity images in (a) and (b). Fitting a mixture of two normal distributions to the bimodal histograms show a greater separation between the peaks in MISS than in DPM.